Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits
G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believ...
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description | G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gαi/o interaction. We investigated the mechanisms of regulation of GIRK by Gαi/o using wild-type Gαi3 (Gαi3WT) and Gαi3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gαi3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gαi3xFP (Gαi3NT) closely mimicked all aspects of Gαi3WT regulation except for a weaker regulation of Ibasal. Gαi3 labeled with YFP within the Gα helical domain preserved regulation of Ibasal but failed to restore fast Ievoked. Titrated expression of Gαi3NT and Gαi3WT confirmed that regulation of Ibasal and of the kinetics of Ievoked of GIRK1/2 are independent functions of Gαi. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gαi3. Thus, Gαi/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gαi/o actively regulates GIRK. Although regulation of Ibasal is a function of GαiGDP, our new findings indicate that regulation of kinetics of Ievoked is mediated by GαiGTP. |
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Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gαi/o interaction. We investigated the mechanisms of regulation of GIRK by Gαi/o using wild-type Gαi3 (Gαi3WT) and Gαi3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gαi3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gαi3xFP (Gαi3NT) closely mimicked all aspects of Gαi3WT regulation except for a weaker regulation of Ibasal. Gαi3 labeled with YFP within the Gα helical domain preserved regulation of Ibasal but failed to restore fast Ievoked. Titrated expression of Gαi3NT and Gαi3WT confirmed that regulation of Ibasal and of the kinetics of Ievoked of GIRK1/2 are independent functions of Gαi. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gαi3. Thus, Gαi/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gαi/o actively regulates GIRK. Although regulation of Ibasal is a function of GαiGDP, our new findings indicate that regulation of kinetics of Ievoked is mediated by GαiGTP.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M111.271056</identifier><identifier>PMID: 21795707</identifier><language>eng</language><publisher>9650 Rockville Pike, Bethesda, MD 20814, U.S.A: Elsevier Inc</publisher><subject>Fluorescence Resonance Energy Transfer (FRET) ; G Protein-coupled Receptors (GPCR) ; G Proteins ; Gating ; Ion Channels ; Signal Transduction</subject><ispartof>The Journal of biological chemistry, 2011-09, Vol.286 (38), p.33223-33235</ispartof><rights>2011 © 2011 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2011 by The American Society for Biochemistry and Molecular Biology, Inc. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3026-b85c9857e02009bdef7d0d438e3c11a17bae3ae8ff19609b01e172bef58431c3</citedby><cites>FETCH-LOGICAL-c3026-b85c9857e02009bdef7d0d438e3c11a17bae3ae8ff19609b01e172bef58431c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190912/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190912/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids></links><search><creatorcontrib>Berlin, Shai</creatorcontrib><creatorcontrib>Tsemakhovich, Vladimir A.</creatorcontrib><creatorcontrib>Castel, Ruth</creatorcontrib><creatorcontrib>Ivanina, Tatiana</creatorcontrib><creatorcontrib>Dessauer, Carmen W.</creatorcontrib><creatorcontrib>Keren-Raifman, Tal</creatorcontrib><creatorcontrib>Dascal, Nathan</creatorcontrib><title>Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits</title><title>The Journal of biological chemistry</title><description>G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gαi/o interaction. We investigated the mechanisms of regulation of GIRK by Gαi/o using wild-type Gαi3 (Gαi3WT) and Gαi3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gαi3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gαi3xFP (Gαi3NT) closely mimicked all aspects of Gαi3WT regulation except for a weaker regulation of Ibasal. Gαi3 labeled with YFP within the Gα helical domain preserved regulation of Ibasal but failed to restore fast Ievoked. Titrated expression of Gαi3NT and Gαi3WT confirmed that regulation of Ibasal and of the kinetics of Ievoked of GIRK1/2 are independent functions of Gαi. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gαi3. Thus, Gαi/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gαi/o actively regulates GIRK. Although regulation of Ibasal is a function of GαiGDP, our new findings indicate that regulation of kinetics of Ievoked is mediated by GαiGTP.</description><subject>Fluorescence Resonance Energy Transfer (FRET)</subject><subject>G Protein-coupled Receptors (GPCR)</subject><subject>G Proteins</subject><subject>Gating</subject><subject>Ion Channels</subject><subject>Signal Transduction</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><recordid>eNp1kcFOGzEQhi3UCgLlzNXHVtUGzzobry-VUFpSRKpWNIfeLNs7C0aLHdneoLxJX4MX4ZnYVVqkHjqX0eif_9PYPyFnwKbAxOz83tjpNwCYlgJYNT8gE2A1L3gFv96QCWMlFLKs6iNynNI9G2om4ZAclSBkJZiYkN_rx0A_u5Sdt5lepA3anGho6SL0m875W2owPyJ6unx-cvRHDBmdp9o3dPl3KrTNbqszNvT6I13cae-xo--XVzfXH-gNblF3g2R29LLrQ8Rk0eduR1fa4CiMYP5K_tmb3ruc3pG3re4Snv7pJ2R9-WW9-Fqsvi-vFherwnJWzgtTV1bWlUBWMiZNg61oWDPjNXILoEEYjVxj3bYg58MCAwRRGmyresbB8hPyaY_d9OYBm_G0qDu1ie5Bx50K2ql_Fe_u1G3YKg6SSSgHwPkeYGNIKWL76gWmxozUkJEaM1L7jAaH3DtweNbWYVTJOvQWGxeH31dNcP_1vgBMXppw</recordid><startdate>20110923</startdate><enddate>20110923</enddate><creator>Berlin, Shai</creator><creator>Tsemakhovich, Vladimir A.</creator><creator>Castel, Ruth</creator><creator>Ivanina, Tatiana</creator><creator>Dessauer, Carmen W.</creator><creator>Keren-Raifman, Tal</creator><creator>Dascal, Nathan</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>5PM</scope></search><sort><creationdate>20110923</creationdate><title>Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits</title><author>Berlin, Shai ; Tsemakhovich, Vladimir A. ; Castel, Ruth ; Ivanina, Tatiana ; Dessauer, Carmen W. ; Keren-Raifman, Tal ; Dascal, Nathan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3026-b85c9857e02009bdef7d0d438e3c11a17bae3ae8ff19609b01e172bef58431c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Fluorescence Resonance Energy Transfer (FRET)</topic><topic>G Protein-coupled Receptors (GPCR)</topic><topic>G Proteins</topic><topic>Gating</topic><topic>Ion Channels</topic><topic>Signal Transduction</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Berlin, Shai</creatorcontrib><creatorcontrib>Tsemakhovich, Vladimir A.</creatorcontrib><creatorcontrib>Castel, Ruth</creatorcontrib><creatorcontrib>Ivanina, Tatiana</creatorcontrib><creatorcontrib>Dessauer, Carmen W.</creatorcontrib><creatorcontrib>Keren-Raifman, Tal</creatorcontrib><creatorcontrib>Dascal, Nathan</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Berlin, Shai</au><au>Tsemakhovich, Vladimir A.</au><au>Castel, Ruth</au><au>Ivanina, Tatiana</au><au>Dessauer, Carmen W.</au><au>Keren-Raifman, Tal</au><au>Dascal, Nathan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits</atitle><jtitle>The Journal of biological chemistry</jtitle><date>2011-09-23</date><risdate>2011</risdate><volume>286</volume><issue>38</issue><spage>33223</spage><epage>33235</epage><pages>33223-33235</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gαi/o interaction. We investigated the mechanisms of regulation of GIRK by Gαi/o using wild-type Gαi3 (Gαi3WT) and Gαi3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gαi3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gαi3xFP (Gαi3NT) closely mimicked all aspects of Gαi3WT regulation except for a weaker regulation of Ibasal. Gαi3 labeled with YFP within the Gα helical domain preserved regulation of Ibasal but failed to restore fast Ievoked. Titrated expression of Gαi3NT and Gαi3WT confirmed that regulation of Ibasal and of the kinetics of Ievoked of GIRK1/2 are independent functions of Gαi. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gαi3. Thus, Gαi/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gαi/o actively regulates GIRK. Although regulation of Ibasal is a function of GαiGDP, our new findings indicate that regulation of kinetics of Ievoked is mediated by GαiGTP.</abstract><cop>9650 Rockville Pike, Bethesda, MD 20814, U.S.A</cop><pub>Elsevier Inc</pub><pmid>21795707</pmid><doi>10.1074/jbc.M111.271056</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Fluorescence Resonance Energy Transfer (FRET) G Protein-coupled Receptors (GPCR) G Proteins Gating Ion Channels Signal Transduction |
title | Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits |
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